Relating to mutagenesis of nucleic acids

Abstract

PCT No. PCT/GB96/02333 Sec. 371 Date Jul. 6, 1998 Sec. 102(e) Date Jul. 6, 1998 PCT Filed Sep. 19, 1996 PCT Pub. No. WO97/11083 PCT Pub. Date Mar. 27, 1997The invention concerns novel compounds having defined structural formulae and methods of mutating a nucleic acid sequence, the method comprising replicating a template sequence in the presence of a nucleoside triphosphate analogue in accordance with the invention, so as to form non-identical copies of the template sequence comprising one or more nucleoside triphosphate analogue residues, and a kit for use in performing the method of the invention.

We claim: 
     
       1. A compound having the structure: ##STR4## where X 1  is selected from the group consisting of O, S, N-alkyl, N +  -dialkyl and N-benzyl; X 2  is selected from the group consisting of triphosphate (P 3  O 9 ) 4- , diphosphate (P 2  O 6 ) 3-  and thiotriphosphate (P 3  O 8  S) 4-  ; and X 3  is selected from the group consisting of H, NH 2 , F and OR, where R is H, methyl, allyl or alkaryl. 
     
     
       2. A compound according to claim 1, wherein X 1  is O. 
     
     
       3. A compound according to claim 2, wherein, X 2  is triphosphate, and X 3  is H or OH. 
     
     
       4. A method of mutating a nucleic acid sequence, comprising replicating a template sequence in the presence of a nucleoside triphosphate analogue in accordance with claim 1, so as to form non-identical copies of the template sequence having one or more nucleoside phosphate analogue residues. 
     
     
       5. A method according to claim 4, comprising replicating a template sequence in the presence of deoxyP triphosphate, so as to form non-identical copies of the template sequence having one or more dP nucleotide residues. 
     
     
       6. A method according to claim 4, further comprising wherein the template sequence is replicated in the presence of one or more additional nucleoside triphosphates. 
     
     
       7. A method according to claim 4, further comprising wherein the template sequence is replicated in the presence of a second compound having the structure: ##STR5## where Y 1  is selected from the group consisting of OH, O-alkyl, NH 2  and N(Alkyl) 2  ; Y 2  is selected from the group consisting of H and NH 2  ; Y 3  is selected from the group consisting of triphosphate (P 3  O 9 ) 4- , diphosphate (P 2  O 6 ) 3-   and thiotriphosphate (P 3  O 8  S) 4-  ; and Y 4  is selected from the group consisting of H, NH 2 , F and OR, where R is H, methyl, allyl or alkaryl. 
     
     
       8. A method according to claim 4, further comprising wherein the template sequence is replicated in the presence of at least one member of the group consisting of 2'-deoxy-8-hydroxyguanosine 5'-triphosphate, 2-amino-9-(2-deoxy-β-D-erythropentofuranosyl)-6-methoxyaminopurine 5'-triphosphate, and O 2  -ethylthymidine triphosphate. 
     
     
       9. A method according to claim 4, further comprising the step of replicating the non-identical copies of the template sequence in the presence of the four normal dNTPs, but in the absence of analogues thereof, to form further non-identical copies of the template sequence comprising only the four normal deoxynucleotides. 
     
     
       10. A method according to claim 4, wherein the replication of the template sequence, and/or the replication of the non-identical copies thereof, is achieved by means of PCR. 
     
     
       11. A method according to claim 4, further comprising wherein the template sequence is replicated in the additional presence of the four normal deoxynucleotides. 
     
     
       12. A method according to claim 5, wherein the template sequence is replicated in the presence of 1 μM to 600 μM 6-(2-deoxy-β-D-erythropentofuranosyl)-3,4 dihydro-8H-pyrimido [4,5-c][1,2]oxazine-7-one 5' triphosphate. 
     
     
       13. A method according to claim 5, wherein the template sequence is replicated in the presence of 1 μM to 600 μM 2'-deoxy-8-hydroxyguanosine 5'-triphosphate. 
     
     
       14. A kit for performing the method of claim 4, comprising said nucleoside triphosphate analogue means for replicating a template sequence so as to incorporate the nucleoside monophosphate portion of the nucleoside triphosphate analogue into non-identical copies of the template sequence, and instructions for use according to said method. 
     
     
       15. A kit according to claim 14, wherein the nucleoside triphosphate analogue is 6-(2-deoxy-β-D-erythropentofuranosyl)-3,4 dihydro-8H-pyrimido [4,5-c][1,2]oxazine-7-one 5'triphosphate. 
     
     
       16. A kit according to claim 14, further comprising wherein the means for replicating the template sequence comprises means for performing the polymerase chain reaction. 
     
     
       17. A kit according to claim 14, further comprising the four normal deoxynucleotides. 
     
     
       18. A kit according to claim 14, further comprising at least one member of the group consisting of 2'-deoxy-8-hydroxyguanosine 5'-triphosphate, 2-amino-9-(2-deoxy-β-D-erythropentofuranosyl)-6-methoxyaminopurine 5'-triphosphate, and O 2  -ethylthymidine triphosphate.